Membrane Phospholipids: The Key Regulator of Tissue Factor Encryption/Decryption

 描述(申请人提供)：凝血因子VIIa(FVIIa)与其细胞表面受体组织因子(Tf)结合启动凝血级联反应。组织因子对于止血是必不可少的，但组织因子的异常表达或激活会导致血栓形成，这是急性心肌梗死、不稳定心绞痛和缺血性卒中的诱发事件。它还会导致炎症和癌症。因此，适当地调节TF的表达和活性不仅对维持止血平衡至关重要，而且对整体健康也是至关重要的。有趣的是，尽管与FVIIa形成了复合体，但细胞表面的大部分Tf以一种隐蔽的状态存在，即没有或几乎没有凝血活性。隐匿性转铁蛋白需要刺激才能成为促凝血活性形式。多种细胞变化将隐匿性因子转化为凝血活性因子。我们最近的研究表明，脂质过氧化产生的最丰富和最具生物活性的物种4-羟基壬烯醛(HNE)可以激活单核细胞和血管内皮细胞中的转铁蛋白。目前，凝血剂活性转铁蛋白与转铁蛋白加密和解密所涉及的隐秘形式或机制有何不同尚不清楚。目前尚不清楚质膜外叶中的磷脂是否在维持转铁蛋白的隐匿状态中起关键作用。在所有已提出的机制中，细胞激活后在质膜外叶的阴离子磷脂外化似乎是隐型转铁蛋白转化为活性转铁蛋白的主要机制。然而，其他机制，如蛋白质二硫键异构酶(PDI)介导的硫醇-二硫键交换反应，也可能在某些细胞类型的TF激活中发挥重要作用。在幼稚细胞中维持转铁蛋白处于隐蔽状态的机制以及在病理生理相关刺激下暴露阴离子磷脂的分子途径尚不清楚。以下具体目标旨在填补这些关于调节细胞表面转铁蛋白活性的知识空白。目的1：验证一种新的假说，即质膜外层小叶中高含量的鞘磷脂是导致转铁蛋白在细胞表面保持隐蔽状态的原因。目的2：阐明磷脂酰丝氨酸外化和HNE激活TF的信号机制(S)，并确定PDI介导的硫醇-二硫键交换通路和脂筏完整性在此过程中的作用。为了加强在细胞系统中的观察，并更好地了解细胞膜脂质如何影响转铁蛋白活性，我们还将对定义的脂质体中加入纯化的转铁蛋白进行额外的研究。从拟议的研究中获得的数据将为理解细胞表面转铁蛋白活性的调节提供新的见解。总体而言，从拟议的研究中获得的知识将有助于了解血栓性疾病的发病机制，并将有助于设计更好的血栓性和出血性疾病的治疗策略。

 DESCRIPTION (provided by applicant): The coagulation cascade is initiated by binding of coagulation factor VIIa (FVIIa) to its cell surface receptor, tissue factor (TF). Tissue factor is essential for hemostasis, but the aberrant expression or activation of TF leads to thrombosis, the precipitating event in acute myocardial infarction, unstable angina, and ischemic stroke. It also contributes to inflammation and cancer. Therefore, the proper regulation of TF expression and the activity is critical for not only to maintenance of the hemostatic balance, but also for health in general. Interestingly, the majority of TF on cell surfaces exists in a cryptic state, i.., with no or little coagulant activity, despite forming complex with FVIIa. A stimulus is required fo cryptic TF to become procoagulant active form. A variety of cellular alterations transforms cryptic TF to coagulant active TF. Our recent studies show that 4- hydoxynonenal (HNE), one of the most abundant and bioactive species produced by the lipid peroxidation, activates TF in monocytic and endothelial cells. At present, it is unclear how the coagulant active TF differs from the cryptic form or mechanics involved in TF encryption and decryption. It is unknown, at present, whether phospholipids present in the outer leaflet of plasma membrane play a critical role in maintaining TF in the cryptic state. Of all the proposed mechanisms, externalization of anionic phospholipids at the outer leaflet of plasma membrane following cell activation appears to be the main mechanism for transformation of the cryptic TF into active TF. However, other mechanisms, such as protein disulfide isomerase (PDI)-mediated thiol-disulfide exchange reactions, may also play an important role in TF activation in certain cell types. Mechanisms responsible for maintaining TF in a cryptic state in naive cells and molecular pathways responsible for exposure of anionic phospholipids in response to pathophysiologically relevant stimuli are unknown. The following specific aims are designed to fill these gaps of the knowledge on the regulation of TF activity at the cell surface. Aim 1: Test a novel hypothesis that high sphingomyelin content in the outer leaflet of the plasma membrane is responsible for maintaining TF in its cryptic state at the cell surface. Here, we will also test that the hydrolysi of sphingomyelin in the plasma membrane plays a key role in TF decryption; Aim 2: Elucidate signaling mechanism(s) involved in externalization of phosphatidylserine and TF activation by HNE, and ascertain contribution of PDI-mediated thiol-disulfide exchange pathways and lipid raft integrity in this process. To strengthen observations made in cell systems, and to have better understanding of how cell membrane lipids influence TF activity, we will also perform additional studies with purified TF incorporated into defined liposomes. The data obtained from the proposed studies will provide new insights into understanding of the regulation of TF activity on cell surfaces. Overall, the knowledge gained from the proposed studies will be helpful in understanding the pathogenesis of thrombotic disorders and will be useful in designing better treatment strategies for both thrombotic and hemorrhagic diseases.